OBJECTIVE To construct a eukaryotic expression vector for RNA interference of the human cyclinD1 gene,and to detect its interference effect in human ovarian cancer cells(HO-8910). METHODS Four target gene segments wer...OBJECTIVE To construct a eukaryotic expression vector for RNA interference of the human cyclinD1 gene,and to detect its interference effect in human ovarian cancer cells(HO-8910). METHODS Four target gene segments were synthesized and cloned into the pSUPER vector respectively to construct four recombinant eukaryotic expression vectors,pSUPER-C1~4.The four recombinant vectors were identified by enzyme digestion analysis and DNAsequencing.Then HO-8910 cells were transfected with the pSUPER-C1~4 vectors and subjected to G418 selection.In G418-resistant cells,the interference effect was detected by RT-PCR. RESULTS Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into the pSUPER vector.The four recombinant vectors inhibited transcription of the cyclinD1 gene.The pSUPER-C2 vector had a better interference effect. CONCLUSION The sequence-specific siRNA effectively interfered with expression of the cyclinD1 gene that was selected.The transcription and expression of the cyclinD1 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the ovarian cancer cel s.These results indicate that it is possible to search for a new tumor gene therapy method.展开更多
AIM: To study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma. METHODS: Paraffin-embedded tissue samples of 25 patients (18 males and 7 females) with hepatocellu...AIM: To study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma. METHODS: Paraffin-embedded tissue samples of 25 patients (18 males and 7 females) with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method (avidin-biotin-peroxidase) for detection of p53, cyclinD1, RB1, c-fos and N-ras proteins. RESULTS: Six (24%), 5 (20%), 12 (48%) and 2 samples (8%) were positive for p53, cyclinD1, C-fos and N-ras expression, respectively. Twenty-two (88%) samples had alterations in the G1 cell-cycle checkpoint protein expression (RB1 or cyclinD1). P53 positive samples showed a higher (9 times) risk of being positive for RB1 protein than p53 negative samples. Loss of expression of RB1 in association with p53 over-expression was observed in 4 (66.7%) of 6 samples. Loss of expression of RB1 was seen in all cyclinD1 positive, 20 (90.9%) N-ras negative, and 11 (50%) C-fos positive samples, respectively. CyclinD1 positive samples showed a higher (2.85 and 4.75 times) risk of being positive for c-fos and N-ras expression than cyclinD1 negative samples. CONCLUSION: The expression of p53, RB1 and c-fos genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.展开更多
文摘OBJECTIVE To construct a eukaryotic expression vector for RNA interference of the human cyclinD1 gene,and to detect its interference effect in human ovarian cancer cells(HO-8910). METHODS Four target gene segments were synthesized and cloned into the pSUPER vector respectively to construct four recombinant eukaryotic expression vectors,pSUPER-C1~4.The four recombinant vectors were identified by enzyme digestion analysis and DNAsequencing.Then HO-8910 cells were transfected with the pSUPER-C1~4 vectors and subjected to G418 selection.In G418-resistant cells,the interference effect was detected by RT-PCR. RESULTS Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into the pSUPER vector.The four recombinant vectors inhibited transcription of the cyclinD1 gene.The pSUPER-C2 vector had a better interference effect. CONCLUSION The sequence-specific siRNA effectively interfered with expression of the cyclinD1 gene that was selected.The transcription and expression of the cyclinD1 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the ovarian cancer cel s.These results indicate that it is possible to search for a new tumor gene therapy method.
文摘AIM: To study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma. METHODS: Paraffin-embedded tissue samples of 25 patients (18 males and 7 females) with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method (avidin-biotin-peroxidase) for detection of p53, cyclinD1, RB1, c-fos and N-ras proteins. RESULTS: Six (24%), 5 (20%), 12 (48%) and 2 samples (8%) were positive for p53, cyclinD1, C-fos and N-ras expression, respectively. Twenty-two (88%) samples had alterations in the G1 cell-cycle checkpoint protein expression (RB1 or cyclinD1). P53 positive samples showed a higher (9 times) risk of being positive for RB1 protein than p53 negative samples. Loss of expression of RB1 in association with p53 over-expression was observed in 4 (66.7%) of 6 samples. Loss of expression of RB1 was seen in all cyclinD1 positive, 20 (90.9%) N-ras negative, and 11 (50%) C-fos positive samples, respectively. CyclinD1 positive samples showed a higher (2.85 and 4.75 times) risk of being positive for c-fos and N-ras expression than cyclinD1 negative samples. CONCLUSION: The expression of p53, RB1 and c-fos genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.
